The present invention, in some embodiments thereof, relates to insulin-producing cells derived from pluripotent stem cells, and methods of generating same.
In type I diabetes, the insulin producing cells, or beta (β)-cells in the islets of Langerhans, are destroyed. Islets of Langerhans are specialized cell aggregates constituting the endocrine pancreas, including β-cells producing insulin (about 55% of the endocrine pancreas in humans), α-cells producing glucagon (about 35% in humans), δ-cells producing somatostatin (3-10%), PPcells producing pancreatic polypeptides (3-5%), and ε-cells producing grehlin (less than 1%). Insulin and glucagon are major regulators of blood glucose levels. In response to high glucose levels, insulin stimulates the uptake of glucose by body cells, fat, liver and muscle cells in particular, where it is converted into energy or stored into fat and glycogen, and therefore lowers the blood glucose level. Glucagon, conversely, stimulates the release of glucose from fat and from glycogen stores in situations of hypoglycemia.
Type I diabetes patients are dependent on injections of insulin to lower their blood glucose level. However, over years, the poor coordination between blood glucose levels and insulin levels often leads to severe deterioration of the patient's health. The physiological regulation of blood glucose as well as general health of such patient can be very much improved by the transplantation of human islets from cadavers. However the need for such transplants is much larger than availability of islet cells from cadaveric donors. In fact only a few thousand transplantations can be done worldwide every year for a potential number of 15 million patients who could benefit from such treatment. There is therefore a need for additional sources of pancreatic islet cells.
Stem cells have been proposed as one such additional source.
For example, the epithelium of the pancreatic duct serves as a source of cells capable of islet neogenesis in the adult, and may constitute the pancreatic stem cells, from which normal renewal of islets occurs throughout life. However, the use of these cells as a source for generation of insulin-producing cells is limited by their low expansion capacity in tissue culture and slow differentiation rate into insulin-producing cells.
Recent studies have shown that tissue stem cells are capable of reprogramming using dominant genes which activate a cascade of developmental events. Thus, mouse [Ferber S. et al. (2000). Nat. Med. 6: 568-572] and Xenopus [Horb M E. Et al., (2003), Curr. Biol. 13: 105-115] liver cells, as well as rat enterocytes [Kojima H et al. (2002), Diabetes 51: 1398-1408] were shown to activate β-cell gene expression following the expression of pancreatic duodenal homeobox 1 (Pdx1), a homeobox factor which plays key roles in pancreas development and gene expression in mature β cells [Jonsson J. et al., (1994) Nature 371: 606-609].
In addition, cultured human fetal liver cells modified by the expression of the Pdx1 gene were shown to produce and store mature insulin in significant amounts, release it in response to physiological glucose levels and replace β-cell function in streptozotocin (STZ)-diabetic non-obese diabetic severe combined immunodeficiency (NOD-scid) mice [Zalzman M. et al., (2003). Proc Natl Acad Sci USA 100: 7253-7258]. These cells expressed multiple β-cell genes, as well as genes of other islet cells and the exocrine pancreas, but continued to express some hepatic genes.
Human embryonic stem cell (hES), established as permanent cell lines from pluripotent human blastocyst inner cell mass, are capable of almost unlimited proliferation in vitro. In vitro, these cells are able to transit through early stages of embryonic development, including all pancreatic lineages. They are the potential source of huge amounts of transplantable donor cells needed for tissue regeneration. The ability to differentiate hESCs into beta-cells highlights a promising strategy to beta-cells replacement [Bernardo et al., 2009, Stem cells (Dayton, Ohio) 27, 341-351; D'Amour et al., 2006, Nature biotechnology 24, 1392-1401; Eshpeter et al., 2008, Cell proliferation 41, 843-858; Jiang et al., 2007, Stem cells (Dayton, Ohio) 25, 1940-1953; Kroon et al., 2008, Nature biotechnology 26, 443-452; Zhang et al., 2009, Cell research 19, 429-438, Sulzbacher et al, 2009, Stem Cell Rev, 5: 159-173].
U.S. Patent Application 20100255580 teaches methods of differentiating pluripotent stem cells towards the pancreatic lineage. However, up until presently directed differentiation of embryonic stem cells has generated cells that only produce low amounts of insulin, compared to beta cells. Therefore, there still remains a significant need to develop conditions for establishing a method of generating insulin-producing cells derived from pluripotent stem cells.